Project 2 Abstract Immune checkpoint inhibitors (ICIs) are providing durable clinical responses in about 20% of cancer patients. But these agents have minimal effect in cancers without intratumoral T cells. Approaches that turn currently unresponsive cancers into ones that are more ?antigenic? are needed to sensitize tumors to ICIs. Tumor genome mutations can express mutant proteins that are tumor-specific and not expressed on normal cells (neoantigens), and cancers with the highest mutational burdens are more likely to respond to single agent ICIs. However, most cancers have lower mutational loads resulting in lower antigenicity, weaker endogenous T cell repertoires, and T cells in the tumor. The best example of a high mutation load cancer is mismatch repair deficient (MMRd) cancers; these cancers often have >1000 mutations/exome and have a >50% response to anti-PD-1 ICIs. But cancers including pancreatic ductal adenocarcinoma (PDA) and microsatellite stabile colorectal carcinoma (MSS CRC) have on average only 50-70 expressed mutations per exome and do not respond to single agent ICIs. Emerging data suggest that it should be possible to develop approaches that combine a neo-antigen targeting vaccine to activate and expand the limited repertoire of T cells specific for the expressed neo-antigens found in low mutation cancers, with ICIs to induce clinically relevant anti-tumor responses. But challenges to successful immunization include knowledge about the repertoire and functional state of pre-existing anti-tumor T cells, identification of the best adjuvants, and approaches that more precisely predict which expressed neo-antigens are the best T cell targets for immunization. We hypothesize that neoantigens are attractive vaccine targets that can raise T cells to be available for further activation by ICIs. Aim 1: we will determine baseline prevelance of pre-existing neo- epitope recognizing T cells using a novel T cell functional assay we developed - Mutation Associated Neoantigen Functional Expansion of Specific T cells (MANAFEST). Aim 2: we will evaluate neo- antigen targeted combination immunotherapy in PDA patients. Aim 3: we will evaluate neo-antigen targeted combination immunotherapy in MSS CRC patients. In aims 2 and 3, we will assess vaccine induced neo-epitope responses in blood and tumor using ELISPOT, multi-plex immunohistochemistry, and MANAFEST. These studies will identify mechanisms of vaccine induced T cell responses. The MANAFEST assay may be used in the future to predict neo-epitopes for vaccine preparation.